[Research progress of genetic research on Char syndrome].

Char syndrome is a rare autosomal dominant genetic disorder characterized by patent ductus arteriosus, facial dysmorphism, and dysplasia of fingers/toes. It may also be associated with multiple papillae, dental dysplasia, and sleep disorders. TFAP2B has proven to be a pathogenic gene for neural crest derivation and development, and several variants of this gene have been identified. Bone morphogenetic protein signaling plays an important role in embryonic development by participating in limb growth and patterning, and regulation of neural crest cell development. TFAP2B is an upstream regulatory gene for bone morphogenetic proteins 2 and 4. Variants of the TFAP2B gene may lead to abnormal proliferation of neural crest cells by affecting the expression of bone morphogenetic proteins, resulting in multiple organ dysplasia syndrome. In addition, TFAP2B variants may only lead to patent ductus arteriosus instead of typical Char syndrome.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia.

Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27-/- mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects.

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient's mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient's mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats.

BACKGROUND: Acellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI-NCSC-SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. METHODS: The effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI-NCSC-SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. RESULTS: (1) EPI-NCSC-SCLCs had good compatibility with ANAs in vitro. (2) In the ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI-NCSC-SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). CONCLUSIONS: EPI-NCSC-SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI-NCSC-SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.

Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds.

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

p53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model.

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

A biased random walk approach for modeling the collective chemotaxis of neural crest cells.

Collective cell migration is a multicellular phenomenon that arises in various biological contexts, including cancer and embryo development. 'Collectiveness' can be promoted by cell-cell interactions such as co-attraction and contact inhibition of locomotion. These mechanisms act on cell polarity, pivotal for directed cell motility, through influencing the intracellular dynamics of small GTPases such as Rac1. To model these dynamics we introduce a biased random walk model, where the bias depends on the internal state of Rac1, and the Rac1 state is influenced by cell-cell interactions and chemoattractive cues. In an extensive simulation study we demonstrate and explain the scope and applicability of the introduced model in various scenarios. The use of a biased random walk model allows for the derivation of a corresponding partial differential equation for the cell density while still maintaining a certain level of intracellular detail from the individual based setting.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

Cornelia de Lange syndrome (CdLS) is a congenital disorder featuring facial dysmorphism, postnatal growth deficits, cognitive disability and upper limb abnormalities. CdLS is genetically heterogeneous, with cases arising from mutation of BRD4, a bromodomain protein that binds and reads acetylated histones. In this study, we have modeled CdLS facial pathology through mouse neural crest cell (NCC)-specific mutation of BRD4 to characterize cellular and molecular function in craniofacial development. Mice with BRD4 NCC loss of function died at birth with severe facial hypoplasia, cleft palate, mid-facial clefting and exencephaly. Following migration, BRD4 mutant NCCs initiated RUNX2 expression for differentiation to osteoblast lineages but failed to induce downstream RUNX2 targets required for lineage commitment. BRD4 bound to active enhancers to regulate expression of osteogenic transcription factors and extracellular matrix components integral for bone formation. RUNX2 physically interacts with a C-terminal domain in the long isoform of BRD4 and can co-occupy osteogenic enhancers. This BRD4 association is required for RUNX2 recruitment and appropriate osteoblast differentiation. We conclude that BRD4 controls facial bone development through osteoblast enhancer regulation of the RUNX2 transcriptional program.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.

Effect of Metformin on Epidermal Neural Crest Stem Cells and Their Potential Application in Ameliorating Paclitaxel-induced Neurotoxicity Phenotype.

AIMS: Epidermal Neural Crest Stem Cells (EPI-NCSCs) have emerged as prospective ideal candidates to meet the fundamental requirements of cell-based therapies in neurodegenerative disorders. The present study aimed to identify the potential of metformin in driving EPI-NCSCs to neuronal/glial differentiation and express neurotrophic factors as well as assess their therapeutic potential for mitigating the main behavioral manifestations of chemotherapy-induced neurotoxicity (CIN). MAIN METHODS: EPI-NCSCs were extracted from the bulge region of hair follicle. Following expansion, transcript and protein expression profiles of key markers for stemness (Nestin, EGR-1, SOX-2 and 10), neurotrophic activity (BDNF, GDNF, NGF, FGF-2, and IL-6), and neuronal (TUB3, DCX, NRF and NeuN) and glial (PDGFRα, NG2, GFAP, and MBP) differentiation were determined on days 1 and 7 post-treatment with 10 and 100 µM metformin using real time-PCR and immunocytochemistry methods. Then, the in vivo function of metformin-treated stem cells was evaluated in the context of paclitaxel CIN. To do so, thermal hyperalgesia, mechanical allodynia, and spatial learning and memory tests were evaluated by Hotplate, Von Frey, and Morris water maze tests. KEY FINDINGS: Our result indicated that exposure of EPI-NCSCs to metformin was associated with progressive decline in stemness markers and enhanced expression levels of several neurotrophic, neuron and oligodendrocyte-specific markers. Further, it was observed that intranasal metformin-treated EPI-NCSCs improved the cognitive impairment, and mechanical and thermal hypersensitivity induced by paclitaxel in rats. SIGNIFICANCE: Collectively, we reasoned that metformin pretreatment of EPI-NCSCs might further enhance their therapeutic benefits against CIN.

Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells.

Periodontal ligament (PDL) stem-like cells (PDLSCs) are promising for regeneration of the periodontium because they demonstrate multipotency, high proliferative capacity, and the potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability. Since PDLSCs are derived from neural crest cells (NCs) and NCs persist in adult PDL tissue, we devised to promote the regeneration of the periodontium by activating NCs to differentiate into PDLSCs. SK-N-SH cells, a neuroblastoma cell line that reportedly has NC-like features, seeded on the extracellular matrix of PDL cells for 2 weeks, resulted in the significant upregulation of PDL marker expression. SK-N-SH cell-derived PDLSCs (SK-PDLSCs) presented phenotypic characteristics comparable to induced pluripotent stem cell (iPSC)-derived PDLSCs (iPDLSCs). The expression levels of various hyaluronic acid (HA)-related genes were upregulated in iPDLSCs and SK-PDLSCs compared with iPSC-derived NCs and SK-N-SH cells, respectively. The knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while low-molecular HA (LMWHA) induction enhanced SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells could be applied as a new model to induce the differentiation of NCs into PDLSCs and that the LMWHA-CD44 relationship is important for the differentiation of NCs into PDLSCs.

KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions, and their impairment causes aplasia cutis.

Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a long-standing enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we found that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss and that disease mutations are dominant negative, resulting in lack of KCTD1/KCTD15 function. We demonstrated that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes resulted in abnormal skin appendages but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells resulted in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells, giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 complexes as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

RET enhancer haplotype-dependent remodeling of the human fetal gut development program.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.

A novel mouse model of intestinal neuronal dysplasia: visualization of the enteric nervous system.

PURPOSE: Intestinal neuronal dysplasia (IND) is a congenital anomaly affecting gastrointestinal neural innervation, but the pathogenesis remains unclear. The homozygous Ncx/Hox11L.1 knockout (Ncx-/-) mice exhibit megacolon and enteric ganglia anomalies, resembling IND phenotypes. Sox10-Venus transgenic mouse were used to visualize enteric neural crest cells in real time. This study aims to establish a novel mouse model of Sox10-Venus+/Ncx-/- mouse to study the pathogenesis of IND. METHODS: Sox10-Venus+/Ncx-/- (Ncx-/-) (n = 8) mice and Sox10-Venus+/Ncx+/+ controls (control) (n = 8) were euthanized at 4-5 weeks old, and excised intestines were examined with fluorescence microscopy. Immunohistochemistry was performed on tissue sections with neural marker Tuj1. RESULTS: Ncx-/- mice exhibited dilated cecum and small intestine. Body weight of Ncx-/- mice was lower with higher ratio of small intestine length relative to body weight. The neural network (Sox10-Venus) was observed along the intestine wall in Ncx-/- and control mice without staining. Ectopic and increased expression of Tuj1 was observed in both small intestine and proximal colon of Ncx-/- mice. CONCLUSION: This study has established a reliable animal model that exhibits characteristics similar to patients with IND. This novel mouse model can allow the easy visualization of ENS in a time- and cost-effective way to study the pathogenesis of IND.

The impact of the recipient intestinal site on the differentiation of transplanted enteric neural crest cells.

PURPOSE: It has long been established that the failure of enteric neural crest cells (ENCCs) to colonize the entire gut results in aganglionosis at the distal colon in Hirschsprung disease (HD). However, it is still unclear how the intestinal microenvironment of the distal aganglionic gut differs from that of the proximal ganglionic gut in HD versus normal gut. We have recently succeeded in transplanting ENCC into aganglionic gut in endothelin receptor B (Ednrb) knockout (KO) mice. to advance the development of cell therapy for HD, it is essential to determine if the transplanted ENCCs differentiate normally in aganglionic gut. Therefore, we designed this study to investigate the impact of the environment of the recipient intestinal tract, at various sites of aganglionic gut, on the differentiation of transplanted ENCCs. METHODS: ENCCs were isolated from Sox10 Venus transgenic (Tg) mouse gut on embryonic day 18.5 (E18.5) and neurospheres (NS) were generated. Then, NS were transplanted into aganglionic KO and wildtype (WT) gut that had been transected just distal to the ENCC wavefront (KO-wf: n = 6, WT: n = 7), and into distal KO gut transected at a site equivalent to that of the WT (KO-d: n = 6) on E12.5. ENCC differentiation was evaluated using whole-mount immunohistochemistry with Tuj-1 (neuronal marker) and GFAP (glial marker) antibodies. RESULTS: The transplanted ENCCs migrated to form the myenteric and submucosal plexus in all groups. The ratio of the area of Tuj-1-positive cells/GFAP-positive cells in migrated cells in the recipient gut was found to be significantly lower in KO-d compared to KO-wf and WT, while there was no significant difference between KO-wf and WT groups. This suggests that neuronal/glial differentiation was decreased in KO-d compared to that in KO-wf and WT groups. CONCLUSION: Our study highlights the differences in ENCC differentiation depending on the site of transplantation. To further develop cell therapy for HD, it is important to consider the impact of the recipient intestinal environment on transplanted ENCCs.